Ex Vivo Chromosomal Radiosensitivity Testing in Patients with Pathological Germline Variants in Breast Cancer High-Susceptibility Genes BReast CAncer 1 and BReast CAncer 2.

BACKGROUND: Individual radiosensitivity is an important factor in the occurrence of undesirable consequences of radiotherapy. The potential for increased radiosensitivity has been linked to highly penetrant heterozygous mutations in DNA repair genes such as BRCA1 and BRCA2. By studying the chromosomal radiosensitivity of BRCA1/2 mutation carriers compared to the general population, we study whether increased chromosomal radiation sensitivity is observed in patients with BRCA1/2 variants. METHODS: Three-color-fluorescence in situ hybridization was performed on ex vivo-irradiated peripheral blood lymphocytes from 64 female patients with a heterozygous germline BRCA1 or BRCA2 mutation. Aberrations in chromosomes #1, #2 and #4 were analyzed. Mean breaks per metaphase (B/M) served as the parameter for chromosomal radiosensitivity. The results were compared with chromosomal radiosensitivity in a cohort of generally healthy individuals and patients with rectal cancer or breast cancer. RESULTS: Patients with BRCA1/2 mutations (n = 64; B/M 0.47) overall showed a significantly higher chromosomal radiosensitivity than general healthy individuals (n = 211; B/M 0.41) and patients with rectal cancer (n = 379; B/M 0.44) and breast cancer (n = 147; B/M 0.45) without proven germline mutations. Chromosomal radiosensitivity varied depending on the locus of the BRCA1/2 mutation. CONCLUSIONS: BRCA1/2 mutations result in slightly increased chromosomal sensitivity to radiation. A few individual patients have a marked increase in radiation sensitivity. Therefore, these patients are at a higher risk for adverse therapeutic consequences.

G2/M Checkpoint Abrogation With Selective Inhibitors Results in Increased Chromatid Breaks and Radiosensitization of 82-6 hTERT and RPE Human Cells.

While technological advances in radiation oncology have led to a more precise delivery of radiation dose and a decreased risk of side effects, there is still a need to better understand the mechanisms underlying DNA damage response (DDR) at the DNA and cytogenetic levels, and to overcome tumor resistance. To maintain genomic stability, cells have developed sophisticated signaling pathways enabling cell cycle arrest to facilitate DNA repair via the DDR-related kinases and their downstream targets, so that DNA damage or DNA replication stress induced by genotoxic therapies can be resolved. ATM, ATR, and Chk1 kinases are key mediators in DDR activation and crucial factors in treatment resistance. It is of importance, therefore, as an alternative to the conventional clonogenic assay, to establish a cytogenetic assay enabling reliable and time-efficient results in evaluating the potency of DDR inhibitors for radiosensitization. Toward this goal, the present study aims at the development and optimization of a chromosomal radiosensitivity assay using the DDR and G2-checkpoint inhibitors as a novel modification compared to the classical G2-assay. Also, it aims at investigating the strengths of this assay for rapid radiosensitivity assessments in cultured cells, and potentially, in tumor cells obtained from biopsies. Specifically, exponentially growing RPE and 82-6 hTERT human cells are irradiated during the G2/M-phase transition in the presence or absence of Caffeine, VE-821, and UCN-1 inhibitors of ATM/ATR, ATR, and Chk1, respectively, and the induced chromatid breaks are used to evaluate cell radiosensitivity and their potency for radiosensitization. The increased yield of chromatid breaks in the presence of DDR inhibitors, which underpins radiosensitization, is similar to that observed in cells from highly radiosensitive AT-patients, and is considered here as 100% radiosensitive internal control. The results highlight the potential of our modified G2-assay using VE-821 to evaluate cell radiosensitivity, the efficacy of DDR inhibitors in radiosensitization, and reinforce the concept that ATM, ATR, and Chk1 represent attractive anticancer drug targets in radiation oncology.

Individual Radiosensitivity in Oncological Patients: Linking Adverse Normal Tissue Reactions and Genetic Features.

Introduction: Adverse effects of radiotherapy (RT) significantly affect patient's quality of life (QOL). The possibility to identify patient-related factors that are associated with individual radiosensitivity would optimize adjuvant RT treatment, limiting the severity of normal tissue reactions, and improving patient's QOL. In this study, we analyzed the relationships between genetic features and toxicity grading manifested by RT patients looking for possible biomarkers of individual radiosensitivity. Methods: Early radiation toxicity was evaluated on 143 oncological patients according to the Common Terminology Criteria for Adverse Events (CTCAE). An individual radiosensitivity (IRS) index defining four classes of radiosensitivity (highly radiosensitive, radiosensitive, normal, and radioresistant) was determined by a G2-chromosomal assay on ex vivo irradiated, patient-derived blood samples. The expression level of 15 radioresponsive genes has been measured by quantitative real-time PCR at 24 h after the first RT fraction, in blood samples of a subset of 57 patients, representing the four IRS classes. Results: By applying univariate and multivariate statistical analyses, we found that fatigue was significantly associated with IRS index. Interestingly, associations were detected between clinical radiation toxicity and gene expression (ATM, CDKN1A, FDXR, SESN1, XPC, ZMAT3, and BCL2/BAX ratio) and between IRS index and gene expression (BBC3, FDXR, GADD45A, and BCL2/BAX). Conclusions: In this prospective cohort study we found that associations exist between normal tissue reactions and genetic features in RT-treated patients. Overall, our findings can contribute to the identification of biological markers to predict RT toxicity in normal tissues.

G2-lymphocyte chromosomal radiosensitivity in patients with LPS responsive beige-like anchor protein (LRBA) deficiency.

Lipopolysaccharide-responsive, beige-like anchor protein (LRBA) deficiency is an autosomal recessive primary immunodeficiency disease characterized by a CVID-like phenotype, particularly severe autoimmunity and inflammatory bowel disease. This study was undertaken to evaluate radiation sensitivity in 11 LRBA-deficient patients. Therefore, stimulated lymphocytes of the studied subjects were exposed to a low dose γ-radiation (100 cGy) in the G2 phase of the cell cycle and chromosomal aberrations were scored. Lymphocytes of age-sex matched healthy individuals used in the same way as controls. Based on the G2-assay, six (54.5%) of the patients had higher radiosensitivity score comparing to the healthy control group, forming the radiosensitive LRBA-deficient patients. This chromosomal radiosensitivity showed that these patients are predisposed to autoimmunity and/or malignancy, and should be protected from unnecessary diagnostic and therapeutic procedures using ionizing radiation and exposure to other DNA damaging agents.

Chromosomal radiosensitivity of triple negative breast cancer patients.

Purpose: Based on clinical and molecular data, breast cancer is a heterogeneous disease. Breast cancers that have no expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) are defined as triple negative breast cancers (TNBCs); luminal cancers have different expressions of ER, PR and/or HER2. TNBCs are frequently linked with advanced disease, poor prognosis and occurrence in young African women, and about 15% of the cases are associated with germline BRCA1/2 mutations. Since radiotherapy is utilized as a principle treatment in the management of TNBC, we aimed to investigate the chromosomal instability and radiosensitivity of lymphocytes in TNBC patients compared to luminal breast cancer patients and healthy controls using the micronucleus (MN) assay. The effect of mutations in breast cancer susceptibility genes on chromosomal radiosensitivity was also evaluated.Methods: Chromosomal radiosensitivity was evaluated in the G0 (83 patients and 90 controls) and S/G2 (34 patients and 17 controls) phase of the cell cycle by exposing blood samples from all patients and controls to 2 and 4 Gy ionizing radiation (IR).Results: In the G0 MN assay, the combined cohort of all breast cancer, TNBC and luminal patients' exhibit significantly elevated spontaneous MN values compared to controls indicating chromosomal instability. Chromosomal radiosensitivity is also significantly elevated in the combined cohort of all breast cancer patients compared to controls. The TNBC patients, however, do not exhibit enhanced chromosomal radiosensitivity. Similarly, in the S/G2 phase, 76% of TNBC patients do not show enhanced chromosomal radiosensitivity compared to the controls. In both the G0 and S/G2 phase, luminal breast cancer patients demonstrate a shift toward chromosomal radiosensitivity compared to TNBC patients and controls.Conclusions: The observations of the MN assay suggest increased chromosomal instability and chromosomal radiosensitivity in South African breast cancer patients. However, in TNBC patients, the irradiated MN values are not elevated. Our results suggest that the healthy lymphocytes in TNBC patients could handle higher doses of IR.

Diagnosis of Fanconi Anaemia by ionising radiation- or mitomycin C-induced micronuclei.

Fanconi Anaemia (FA) is an autosomal recessive disorder characterised by defects in DNA repair, associated with chromosomal instability and cellular hypersensitivity to DNA cross-linking agents such as mitomycin C (MMC). The FA repair pathway involves complex DNA repair mechanisms crucial for genomic stability. Deficiencies in DNA repair genes give rise to chromosomal radiosensitivity. FA patients have shown increased clinical radiosensitivity by exhibiting adverse normal tissue side-effects. The study aimed to investigate chromosomal radiosensitivity of homozygous and heterozygous carriers of FA mutations using three micronucleus (MN) assays. The G0 and S/G2MN assays are cytogenetic assays to evaluate DNA damage induced by ionising radiation in different phases of the cell cycle. The MMC MN assay detects DNA damage induced by a crosslinking agent in the G0 phase. Patients with a clinical diagnosis of FA and their parents were screened for the complete coding region of 20 FA genes. Blood samples of all FA patients and parents were exposed to ionising radiation of 2 and 4Gy. Chromosomal radiosensitivity was evaluated in the G0 and S/G2 phase. Most of our patients were homozygous for the founder mutation FANCG c.637_643delTACCGCC; p.(Tyr213Lysfs*6) while one patient was compound heterozygous for FANCG c.637_643delTACCGCC and FANCG c.1379G > A, p.(Gly460Asp), a novel missense mutation. Another patient was compound heterozygous for two deleterious FANCA mutations. In FA patients, the G0- and S/G2-MN assays show significantly increased chromosomal radiosensitivity and genomic instability. Moreover, chromosomal damage was significantly elevated in MMC treated FA cells. We also observed an increase in chromosomal radiosensitivity and genomic instability in the parents using 3 assays. The effect was significant using the MMC MN assay. The MMC MN assay is advantageous as it is less labour intense, time effective and has potential as a reliable alternative method for detecting FA patients from parents and controls.

Radiation-induced chromosomal instability of human lymphocytes as a marker of breast cancer risk. / Radiacijno-indukovana hromosomna nestabil''nist'' limfocytiv ljudyny jak pokaznyk ryzyku raku grudnoi' zalozy.

OBJECTIVE: to assess the variability of the levels of chromosome aberrations induced by the in vitro irradiation of lymphocytes of breast cancer (BC) patients in comparison with healthy individuals. Materials and methods. Samples of peripheral blood for lymphocyte cultures were obtained from 44 healthy women and 37 primary patients with BC (T1-2N1M0). Lymphocyte X-ray irradiation was carried out at G0 and G2 phase of lymphocyte cell cycle (G0 and G2 assay) with the dose of 1,5 Gy before PHA stimulation in G0 assay and 0,5 Gy - at 47 h of cultivation in G2 assay. Preparations of metaphase chromosomes were made according standard protocols. Results and conclusions. Inter-individual variation of G2 chromosome aberration yields was significantly higher in comparison with G0 chromosomal radiosensitivity in both examined groups. According to G2 assay the fraction of individuals with elevated chromosomal radiosensitivity among healthy women and BC patients was 11.4 and 38 %, respectively. The results obtained support the concept of association between predisposition to BC, radiationinduced G2 chromosomal instability and efficiency of DNA repair activated by cell irradiation in G2 phase. It is assumed that individuals with elevated G2 aberration scores from the control group need further examinations on the BC risk and primary prevention of radiation induced cancer.

Radiation Induced G2 Chromatic Break and Repairs Kinetics in Human Lymphoblastoid Cells / 대한치료방사선과학회지

In understanding radiosensitivity a new concept of inherent radiosensitivity based on individuality and heterogeneity within a population has recently beer explored. There has been some discussion of possible mechanism underlying differences in radiosensitivity between cells. Ataxia telangiectasia(AT), a rare autosomal recessive genetic disorder, is characterized by hypersensitivity to lonizing radiation and other DNA damaging agents at the cellular level. There have been a lot of efforts to describe the cause of this hypersensitivity to radiation. At the cellular level, chromosome repair kinetics study would be an appropriate approach. The purpose of this study was to better understand radiosensitivity in an approach to investigate kinetics of induction and repair of G2 chromatic breaks using normal, AT heterozygous(ATH), and AT homozygous lymphoblastoid cell lines. In an attempt to estimate initial damage, 9-betaD-arabinosyl-2-fluoroadenine, an inhibitor of DNA synthesis and repair, was used in this study. It was found from this study that radiation induces higher chromatid breaks in AT than in normal and ATH cells. There was no significant differences of initial chromatid breaks between normal and ATH cells. Repair kinetics was the same for all. So the higher level of breaks in AT G2 cells is thought to be a reflection of the increased initial damage. The amount of initial damage correlated well with survival fraction at 2 Gy of cell survival curve following radiation. Therefore, the difference of radiosensitivity in terms of G2 chromosomal sensitivity is thought to result from the difference of initial damage.